Real-time reverse transcription loop-mediated isothermal amplification for rapid detection of West Nile virus
Real-time reverse transcription loop-mediated isothermal amplification for rapid detection of West Nile virus
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DOI：
10.1128/jcm.42.1.257-263.2004
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发表时间：
2004-01-01
影响因子：
9.4
通讯作者：
Morita, K
中科院分区：
文献类型：
--
作者：
Parida, M;Posadas, G;Morita, K
建立了一步法、单管、实时加速逆转录环介导等温扩增（RT-LAMP）检测西尼罗病毒（WN）囊膜基因的方法。RT-LAMP检测是一种新的基因扩增方法，其在等温条件下使用一组六种专门设计的引物以高特异性、高效性和快速性扩增核酸，所述引物识别靶的八种不同序列。整个过程是非常简单和快速的，并且在63 ℃下与逆转录酶和Bst DNA聚合酶一起在单管中孵育所有试剂可以在不到1小时内获得扩增。基因扩增的检测可以通过琼脂糖凝胶电泳以及在廉价的浊度计中实时监测来完成。当RT-LAMP测定的灵敏度与常规RT-PCR的灵敏度进行比较时，发现RT-LAMP测定显示出比RT-PCR高10倍的灵敏度，检测极限为0.1PFU的病毒。通过使用实时监测，可以在短短17分钟内检测到104 PFU的病毒。RT-LAMP检测的特异性通过与黄病毒组其他密切相关的成员不存在任何交叉反应来验证，然后进行限制性消化和核酸测序扩增产物。这些结果表明，RT-LAMP检测是非常快速，成本效益，高度敏感，和具体的，并具有潜在的有用性，快速，全面的WN病毒监测沿着病毒分离和/或血清学。
A one-step, single tube, real-time accelerated reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detecting the envelope gene of West Nile (WN) virus. The RT-LAMP assay is a novel method of gene amplification that amplifies nucleic acid with high specificity, efficiency, and rapidity under isothermal conditions with a set of six specially designed primers that recognize eight distinct sequences of the target. The whole procedure is very simple and rapid, and amplification can be obtained in less than I h by incubating all of the reagents in a single tube with reverse transcriptase and Bst DNA polymerase at 63degreesC. Detection of gene amplification could be accomplished by agarose gel electrophoresis, as well as by real-time monitoring in an inexpensive turbidimeter. When the sensitivity of the RT-LAMP assay was compared to that of conventional RT-PCR, it was found that the RT-LAMP assay demonstrated 10-fold higher sensitivity compared to RT-PCR, with a detection limit of 0.1 PFU of virus. By using real-time monitoring, 104 PFU of virus could be detected in as little as 17 min. The specificity of the RT-LAMP assay was validated by the absence of any cross-reaction with other, closely related, members of the Flavivirus group, followed by restriction digestion and nucleotide sequencing of the amplified product. These results indicate that the RT-LAMP assay is extremely rapid, cost-effective, highly sensitive, and specific and has potential usefulness for rapid, comprehensive WN virus surveillance along with virus isolation and/or serology.